Exploiting the order of membrane-associating molecules
Kishore, Anita Indira
MetadataShow full item record
Novel nuclear magnetic resonance (NMR) experiments and computational methods have been developed to characterize membrane-associated molecules. The methodology has been applied to the membrane-associating protein Adenosine diphosphate Ribosylation Factor 1 (ARF1) and its ligands when they are organized at the membrane surface. ARF1 is a 21 kDa GDP-GTP switch protein that interacts reversibly with a membrane through the GTP-controlled exposure of its N-myristoyl chain and N-terminal amphipathic helix. Current structural methods have failed to produce ARF1’s structure and orientation when associated with the membrane, and new methodology is urgently needed to better understand ARF1’s role in vesicle trafficking. NMR is well suited to the study of membrane-associating systems. By taking advantage of the magnetic properties of liquid crystalline model membranes as both bilayered membrane mimetics and orienting media for molecules dissolved within them, non-myristoylated (non-myr) ARF1 and ARF1’s ligands have been uniformly ordered, generating complex NMR spectra rich in structural information. To reduce the complexity, variable angle sample spinning NMR was used to extract phosphorus chemical shift anisotropy offsets from membrane-anchored phosphatidylinositol bisphosphate (PIP2), a signaling lipid believed to promote membrane association of ARF1, yielding the headgroup orientation of PIP2. Nitrogen-proton residual dipolar couplings were also used to ascertain the location and extent of non-myrARF1’s interaction with PIP in a membrane environment. In anticipation of strong membrane association between PIP2 and ARF1, a novel approach based on the observation of carbon chemical shift anisotropy offsets for ARF1-bound nucleotide cofactors (GDP) was also developed. My work suggests that PIP2 orients with its inositol headgroup bent toward the membrane and charged phosphate groups exposed, possibly mediating ARF1’s membrane-associating ability. When combined in a model membrane with PIP, non-myrARF1 did show extensive interaction with the lipid, but PIP2 appeared to be extractedfrom the membrane, possibly by secluding PIP2’s acyl chains within the hydrophobic pocket normally occupied by ARF1’s own myristoyl chain. This work suggests the importance of the myristoyl chain in not only modulating membrane interactions but in regulating more specific interactions with lipid components. The work brings into question many previous conclusions based on studies of the more easily obtained non-myrARF1 and N-terminal truncated forms of ARF1.